Rapid quantification of butyric acid-producing bacteria using real-time PCR

ABSTRACT

A set of primer pairs for amplifying a nucleic acid of a butyric acid-producing bacteria, each primer containing an oligonucleotide selected from the buk gene region of the butyric acid-producing bacteria. The primers are particularly suitable for amplifying the nucleic acid of butyric acid-producing bacteria present in gas and oil production operations.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method for the rapid detection andquantification of butyric acid-producing bacteria using real-timequantitative polymerase chain reactions (qPCR).

2. Description of Related Art

Microbiologically influenced corrosion is a significant problemaffecting the gas and oil industry as well as other industries, and themonitoring of microbial populations within such industries is acomponent of overall corrosion control operations. Traditionally, themonitoring of microbial populations in samples obtained from gas and oilproduction operations has employed microbial growth tests. In suchtests, samples are diluted to various levels and used to inoculatemicrobial growth media that is designed to favor the growth of varioustypes of bacteria. After days to several weeks of incubation, the growthtests are scored based on the presence or absence of growth in thesevarious microbiological media. Unfortunately, as numerous researchesshow, only about 0.1% to about 10% bacteria from environmental samplescan actually grow in an artificial medium, and a significant portion ofbacteria growing in the media are not actually the target bacteria.Therefore, growth tests are unable to provide the accuratequantification of target bacteria in the samples.

To circumvent problems associated with such growth-based methods, manyculture-independent genetic techniques have been developed in the pastdecade and applied for the detection of pathogens and quality controland assurance of products in the medicine and food industries. Becausemany ecosystems, including pipeline systems, have a relatively lowabundance of microorganisms, the polymerase chain reaction (PCR) hasbeen widely used to amplify the genetic signals of microbes in complexenvironmental samples. However, traditional PCR-based methods aresignificantly biased by amplification efficiency and the depletion ofPCR reagents. Real-time quantitative PCR (qPCR) has been developed andused in the last few years in the medical and food research/industriesto detect and quantify a number of pathogenic or infectiousmicroorganisms. Quantitative PCR has also been used to determine theabundance of microorganisms in many different types of complexenvironmental samples such as sediments, water, wastewater, and marinesamples. The advantage of quantitative PCR over traditional PCR is thatit provides more accurate and reproducible quantification ofmicroorganisms because quantitative PCR quantifies PCR products duringthe logarithmic phase of the reactions. Moreover, quantitative PCRoffers a dynamic detection range of six orders of magnitude or more,does not need post-PCR manipulation, and has the capability of highthroughput analysis.

Acid-producing bacteria are present in a variety of environments,including oil- and gas-bearing formations, soils, and domestic,industrial, and mining wastewaters. Acid-producing bacteria produceorganic acids such as acetic, butyric, formatic, lactic, succinic, andpropionic acids, and contribute to metal corrosion.

While the total bacterial number in an oil or gas production sampleindicates the general conditions for bacteria to grow in this uniqueenvironment, and, thus, the potential risk of microbial corrosion, somespecific groups of microorganisms commonly found in such samplesprobably play a more important role in the corrosion process. One suchmicroorganism is a butyric acid-producing bacteria which has been foundto be present in environmental samples and, in particular, samples fromgas and oil production operations.

SUMMARY OF THE INVENTION

Accordingly, it is one object of this invention to provide aquantitative polymerase chain reaction assay that can be used to rapidlydetect and quantify the butyric acid-producing bacteria in environmentalsamples, including, but not limited to, gas and oil productionoperations samples.

The most abundant acid-producing bacteria found in gas and oilproduction environments are acetic acid-producing bacteria and butyricacid-producing bacteria. The gene buk encodes the key enzyme butyratekinase, which along with phosphotransbutyrylase, catalyzes theconversion of butyryl coenzyme A to butyrate. To address theabove-stated object as well as other objects of this invention, areal-time qPCR assay has been developed by targeting the gene bukencoding butyrate kinase in butyric acid-producing bacteria. In oneaspect, this invention features a set of PCR primer pairs that has beencreated to meet the specific requirements of real-time qPCR, whichprimer pairs amplify an approximately 103-bp DNA fragment from the bukgene of butyric acid-producing bacteria. The creation of the specificPCR primers comprising the primer pairs was accomplished by determiningthe DNA sequences of buk genes in butyric acid-producing bacteria mostcommonly found in gas and oil production operations. Each primer of theset of PCR primer pairs contains an oligonucleotide selected from thebuk gene region of the butyric acid-producing bacteria. The set of PCRprimer pairs comprises forward primers, buk-5F1 and buk-5F2, havingoligonucleotides selected from the group of SEQ ID NO: 1 and SEQ ID NO:2, respectively, and reverse primers, buk-6R1, buk-6R2, and buk-6R3,having oligonucleotides selected from the group of SEQ ID NO: 3, SEQ IDNO: 4, and SEQ ID NO: 5, respectively.

The set of PCR primer pairs of this invention has been evaluated in SYBRGreen I real-time qPCR on reference butyric acid-producing bacteriastrains as well as gas pipeline samples. The primer combination givesthe best detection and quantification of butyric acid-producing bacteriacommonly found in the oil and gas production operations. In addition tobeing appropriate for the real-time PCR quantification of butyricacid-producing bacteria, the set of primers of this invention isspecific for samples from gas and oil production operations and combineshigh throughput with high analytical sensitivity and precision, offeringa dynamic detection range of 6 orders of magnitude or more.

In another aspect, this invention features a method for quantifying theamount of butyric acid-producing bacteria in a sample from a gas and/oroil production environment in which a nucleic acid in the genome of thebutyric acid-producing bacteria is amplified by quantitative PCR to forma double-stranded nucleic acid product, the nucleic acid product iscontacted with a label, such as a fluorophore, e.g. SYBR Green I, in asolution for binding therebetween, and the butyric acid-producingbacteria is quantified by monitoring the signal produced by the boundlabel, the intensity of which is a function of the quantity of the bukgene in butyric acid-producing bacteria in the sample.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS Definitions

As used herein, the term “gene” means a DNA sequence containinginformation required for expression of a polypeptide or protein.

As used herein, the term “primer” refers to a single-strandedoligonucleotide that is extended by covalent bonding of nucleotidemonomers during amplification or polymerization of a nucleic acidmolecule.

As used herein, the term “template” refers to a double-stranded orsingle-stranded nucleic acid molecule which is to be amplified,synthesized or sequenced. In the case of a double-stranded DNA molecule,denaturation of its strands to form a first and the second strand isperformed before these molecules may be amplified, synthesized orsequenced. A primer, complementary to a portion of a template ishybridized under appropriate conditions and a polymerase thensynthesizes a molecule complementary to the template or a portionthereof.

As used herein, the term “amplification” refers to any in vitro methodfor increasing the number of copies of a nucleotide sequence with theuse of a DNA polymerase. Nucleic acid amplification results in theincorporation of nucleotides into a DNA molecule or primer, therebyforming a new DNA molecule complementary to a DNA template. The formedDNA molecule and its template can be used as templates to synthesizeadditional DNA molecules. As used herein, one amplification reaction mayconsist of many rounds of DNA replication. DNA amplification reactionsinclude polymerase chain reactions, PCR, which may consist of 10 to 50cycles of denaturization and synthesis of a DNA molecule.

As used herein, the term “oligonucleotide” refers to a synthetic ornatural molecule comprising a covalently linked sequence of nucleotideswhich are joined by a phosphodiester bond between the 3′ position of thepentose of one nucleotide and the 5′ position of the pentose of theadjacent nucleotide.

The invention disclosed herein is a PCR assay targeting the gene bukencoding butyrate kinase in butyric acid-producing bacteria using atleast one of two novel forward primers, SEQ ID NO: 1 and SEQ ID NO: 2,and using at least one of three novel reverse primers, SEQ ID NO: 3, SEQID NO: 4 and SEQ ID NO: 5. The primers were designed based upon multiplealignments of partial sequences of the targeted buk gene retrieved fromGenBank and gas pipeline samples. BioEdit Sequence Alignment Editor,Version 5.0.9, was used for multiple alignments of sequences, and manualsearches for alignments were also conducted to determine candidateprimer sequences. All primers were checked for hairpin structure anddimmers using a free Oligonucleotide Analyzer (available atwww.rnature.com/oligonucleotide.html). MeltCal software, version 2.1 wasthen used to check the cross-hybridization among the sequences ofprimers used in some assay and calculate the melting temperature (Tm)using a nearest neighbor model. The sequences meet the generalrequirements of primer design for real-time quantitative PCR suggestedby the RotoGene Real-Time PCR system manual provided by Corbett Researchor in Inglis, G. D. et al., “Direct Quantification of Campylobacterjejuni and Campylobacter lanienae in Feces of Cattle by Real-TimeQuantitative PCR”, Appl. Environ. Microbiol., 2004. 70(4): pp 2296-2306.In addition, all primer sequences were analyzed for specificity using aBLAST SEARCH FOR SHORT, NEARLY EXACT MATCHES program and were found notto cross-react with any other non-target organisms.

The PCR primer pairs of this invention are based upon the DNA sequencesof buk genes from subsets of butyric acid-producing bacteria mostcommonly found in gas and oil production operations. Thus, the PCRprimer pairs are ideally suited to accurately quantify butyricacid-producing bacteria in gas and oil production operations that haverelevance to microbiologically influenced corrosion. If PCR primers weredesigned based on the DNA sequences of all known buk genes published inDNA sequence databases such as GenBank, the regions of conservedsequences that are suitable for use in designing PCR primers would bemore limited. Primers designed on the entirety of all buk DNA sequenceswould by necessity fail to produce good yields from some buk genes.

Not all species of butyric acid-producing bacteria are commonly presentin gas and oil production environments. Thus, PCR primers that targetthe sequences of those bacteria most commonly encountered in gas and oilproduction environments yield superior results in developing real-timePCR methods to accurately quantify butyric acid-producing bacteria inthese samples and are capable of detecting the target butyricacid-producing bacteria with the highest possible sensitivity. Thequantification using the real-time PCR method and primers of thisinvention is achieved by measuring the fluorescent signal of SYBR-GreenI dye resulting from its specific binding to the double-strandedamplified fragments.

The target butyric acid-producing bacteria for which the set of PCRprimers of this invention are a perfect match include Bacillus anthracisstr. ‘Ames Ancestor’ (AE017334), Bacillus anthracis str. Ames(AE017037), Bacillus anthracis str. Sterne (AE017225), Bacillus cereusATCC 10987 (AE017277), Bacillus cereus ATCC 14579 (AE017011), Bacilluscereus E33L (CP000001), Bacillus subtilis (Z99116), Bacillusthuringiensis serovar konkukian str. 97-27 (AE017355), Clostridiumacetobutylicum ATCC 824 (AE007804), and Clostridium acetobutylicumbutyrate kinase (L14744). All of these bacteria have the buk gene.

In general, the PCR amplification process employed in the method of thisinvention may be carried out using procedures known to those skilled inthe art, namely, denaturing, annealing, and elongation, which can berepeated as many times as necessary to produce the desired amount of thetarget nucleic acid. While the number of cycles may be affected by anyof a number of factors, such as the nature of the sample, the number ofcycles required to achieve the desired amplification in accordance withthis invention is generally in the range of about 10 to about 50.However, cycles numbers outside of this range may be necessary and,thus, the statement of the above stated range is in no way intended tolimit the scope of this invention.

As previously indicated, the set of PCR primers of this inventioncomprises two forward primers, buk-5F1 (5′-CCA TGC ATT AAA TCA AAAAGC-3′, melting temperature (Tm): 56.5° C., SEQ ID NO: 1), buk-5F2(5′-CCA TGC GTT AAA CCA AAA AGC-3′, Tm: 61.1° C., SEQ ID NO: 2), andthree reverse primers buk-6R1 (5′-AGT ACC TCC ACC CAT GTG-3′, Tm: 59.6°C., SEQ ID NO: 3), buk-6R2 (5′-AAT ACC TCC GCC CAT ATG-3′, Tm: 57.5° C.,SEQ ID NO: 4), buk-6R3 (5′-AAT ACC GCC RCC CAT ATG-3′, Tm: 58.1-61.4,SEQ ID NO: 5), all of which are perfect matches to the sequences of theabove-mentioned butyric acid-producing bacteria. Thus, when this set ofPCR primer pairs is used in the PCR amplification, only theabove-mentioned butyric acid-producing bacteria are amplified. Theprimers, when used as pairs (at least one forward primer and at leastone reverse primer), do not falsely amplify any bacteria which is not abutyric acid-producing bacteria. In addition, the primer pairs do notamplify sequences from Eukaryota, a high-level organism.

EXAMPLE

A q-PCR was performed using the primers of this invention, a Rotor-Gene3000, 4 Channel Multiplexing, real-time PCR system (Corbett Research,Sydney, Australia), and a QuantiTect SYBR Green PCR kit (Qiagen, Inc.,Valencia, Calif.). For quantification of the total bacteria, each 20 μLq-PCR reaction contained 1× QuantiTect SYBR Green PCR Master Mix, 800 nMforward primers (400 nM each of buk-5F1 and buk-5F2), 1400 nM reverseprimers (400 nM buk-6R1; 200 nM buk-6R2; and 800 nM buk-6F3), one μL oftemplate, and 4.6 μL water. The cycling conditions consisted of 15minutes of incubation at 95° C. followed by 35 cycles of 95° C. for 30sec, 52° C. for 30 sec, and 71° C. for denaturing, annealing andelongation, respectively, followed by 70° C. for 30 sec, and 70° C. to99° C. to generate melting curves.

Using Clostridium acetobutylicum genomic DNA (ATCC 824) as a template,the linear amplification range for the quantification of the buk genewas 1×10⁷ to 2×10² copies per reaction (correlation coefficient,R²>0.9999). The q-PCR product size was 103 bp from position 459 to 561of Clostridium acetobutylicum ATCC 824 buk gene (complete genome GenBankAccession Number NC_(—)003030, from 3224097 to 3226164).

While in the foregoing detailed description this invention has beendescribed in relation to certain preferred embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein can be varied considerably without departing from the basicprinciples of the invention.

1. A set of primer pairs for amplifying a nucleic acid of a butyricacid-producing bacteria, each primer containing an oligonucleotideselected from the buk gene region of said butyric acid-producingbacteria, wherein the first primer is an oligonucleotide selected fromthe group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 and the secondprimer is an oligonucleotide selected from the group consisting of SEQID NO: 3, SEQ ID NO: 4 and SEQ ID NO:
 5. 2. The primer pair of claim 1,wherein said butyric acid-producing bacteria is selected from the groupconsisting of Bacillus anthracis str. ‘Ames Ancestor’ (Accession NumberAE017334), Bacillus anthracis str. Ames (Accession Number AE017037),Bacillus anthracis str. Sterne (Accession Number AE017225), Bacilluscereus ATCC 10987 (Accession Number AE017277), Bacillus cereus ATCC14579 (Accession Number AE017011), Bacillus cereus E33L (AccessionNumber CP000001), Bacillus subtilis (Accession Number Z99116), Bacillusthuringiensis serovar konkukian str. 97-27 (AE017355), Clostridiumacetobutylicum ATCC 824 (Accession Number AE007804), Clostridiumacetobutylicum butyrate kinase (Accession Number L14744), and mixturesthereof.
 3. A PCR amplification method for quantification of butyricacid-producing bacteria comprising amplifying a nucleic acid of abutyric acid producing bacteria in the presence of forward primersselected from the group consisting of SEQ ID NO: 1 and SEQ ID NO:
 2. andmixtures thereof, reverse primers selected from the group consisting ofSEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 and mixtures thereof.
 4. ThePCR amplification method of claim 3, wherein said forward primers andsaid reverse primers comprise oligonucleotides selected from the bukgene region of said butyric acid-producing bacteria.
 5. The PCRamplification method of claim 3, wherein nucleic acid produced by saidmethod is quantified using a SYBR Green I dye.
 6. The PCR amplificationmethod of claim 3, wherein said butyric acid-producing bacteria isdisposed in an environmental sample.
 7. The PCR amplification method ofclaim 3, wherein said environmental sample is taken from at least one ofa natural gas production operation and an oil production operation.